Confocal tandem scanning reflected light microscope

ABSTRACT

An improved method and apparatus for easily and accurately aligning confocal tandem scanning reflected light microscopes having scanning disc hole diameters as small as 20 microns are described. The method involves the observation and analysis of Moire and other patterns of light occurring within the microscope, and using the apparatus to make very sensitive adjustments that result in a fully aligned, rugged, and stable confocal tandem scanning reflected light microscope offering improved resolution, contrast, and optical slicing capability.

TECHNICAL FIELD

This invention relates to an improved confocal tandem scanning reflectedlight microscope. More specifically, the present invention relates to arugged, stable, easily adjustable apparatus for mounting the majoroptical components, and a new method for aligning confocal tandemscanning reflected light microscopes.

BACKGROUND ART

In U.S. Pat. No. 3,517,980 of Jun. 30, 1970, Petran and Hadravskydescribed a confocal tandem scanning reflected light microscope whereinlight is passed to a target through a rotating disc containing severalthousand very small holes. Such light is then reflected from the targetin a pattern of pinpoints of light through holes in the scanning discthat are exactly opposite the illumination holes. Such a microscopeimproves resolving power and permits observation of objects covered bytranslucent materials. The embodiments produced and available to datehave used scanning discs having hole diameters of about 60 microns,which holes are larger than is desirable for best microscope resolution,contrast discrimination, and thinnest optical slicing capability. Themajor reason for not producing microscopes with smaller disc holes isthe inability to align such microscopes precisely and easily, and tomaintain such alignment, using component mounting schemes and adjustmenttechniques currently embodied.

This present invention relates to confocal tandem scanning reflectedlight microscopes having scanning disc holes of diameter as small as 20microns, and more particularly to an improved optical element mountingapparatus, and to an improved method of adjusting such apparatus.Confocal tandem scanning microscopes having very small scanning discholes are particularly important and useful for real time,high-magnification viewing of, among other things, living bulktranslucent tissues (as in the living eye) and the thinnest possibleoptical slices thereof, with best resolution and contrastdiscrimination, all a direct result of the presence of smaller scanningdisc holes and precise optical path alignment methods.

In current confocal tandem scanning microscopes using 60 micron holes,alignment of optical elements in the light path is critical, difficultand time consuming, and may not be precise enough to obtain bestmicroscope performance, even for the 60 micron holes. In devicescurrently on the market, the various elements to be adjusted arebasically stacked (piggybacked) so that certain adjustments along thestack are inter-related with other adjustments of elements in the stack.The alignment process involves placing external red and green lightsources at the illumination area and viewing area of the discrespectively, and attempting to move the interacting components so as toachieve exact registration of all the red and green pinpoints of light,when viewed through the objective lens opening (both the illuminationand the viewing areas can be seen superimposed due to the presence of abeamsplitter). If adjustments can be made so that all the red holes canbe exactly superimposed on the all the green holes, the microscope iscorrectly aligned. Such a method is very laborious and somewhatimprecise when the holes are 60 microns in diameter, and isprohibitively laborious and very imprecise when the holes are 20 or 30microns in diameter, and therefore is not practical for use when thesmaller holes are employed.

One other type of confocal tandem scanning microscope that is currentlyavailable (called a "one-path" or "one-sided" confocal tandem scanningmicroscope) uses disc holes in the 20 to 40 micron range, and requiresno special alignment, since it projects the illumination and receives itback through the same scanning hole. Such a microscope is less complexand generally less expensive than a full "two-path" microscope as in theoriginal invention, but has the disadvantage of requiring that thebeamsplitter be placed in apposition to the opposite surface of thedisc, so that the illuminating light passes through the beamsplitterbefore passing through the disk, thus exposing the eyepiece to asubstantially larger amount of stray reflections than in the two- pathmicroscope. A one-path embodiment is most effective where the light(signal) reflected from the target is bright relative to the light fromundesired sources (noise), including stray internal reflections. Such amicroscope is readily usable in observing integrated circuit "chips" andother high-reflectance or high contrast targets; however, it is lesseffective where the light reflected from the target is well belowambient light, such as when the target is one of many types of softbiological tissue (e.g., living eyes) or other relatively non-reflectiveor low contrast materials.

SUMMARY AND BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a full two-pathconfocal tandem scanning microscope having scanning disc holes (discholes may be through-holes or may be apertures etched in depositedmaterials, as in chrome deposited on clear glass) in the 20-30 microndiameter range, thereby attaining enhanced capability to opticallyslice, resolve, and discriminate in living, bulk, soft translucenttissue or similar material in real time.

It is a further object of the present invention to describe rugged andstable apparatuses on which are mounted such microscope opticalcomponents, incorporating a method of non-interactive componentarrangement having all necessary (6) degrees of freedom so that accuratealignment of the microscope can be achieved, with all individualadjustments being easily, quickly, and precisely accomplished with aminimum of interactions.

It is a further object of the present invention that the adjustmentprocesses use only the observation and analysis of information availablefrom reflected light patterns within the microscope, thereby eliminatingthe requirement for registration of externally supplied lights, and thehole-size limitations associated therewith.

It is a further objective of the present invention to describe astep-by-step adjusting procedure for achieving best alignment in theconfocal tandem scanning reflected light microscope.

According to the present invention, a tandem scanning reflected lightmicroscope employs a base member (mainframe) on which is mountedsubstantially all of the adjustment members and to which all adjustmentmembers are referenced. In the normal use of the microscope, theobjective lens and the target constitute a cat's-eye retro-reflector.Light from a point on an illumination area as defined by a hole in ascanning disc is focused by the objective to a point on the target. (Seefor instance applicant's co-pending application Ser. No. 07/497,318,filed Mar. 22, 1990 now U.S. Pat. No. 5,083,220 issued Jan. 21, 1992,and assigned to the same assignee as the present invention.) This lightreflects from the target and is focused by the objective back to thepoint from which it came. This is simultaneously true for each disc holein the illumination area as is the normal effect of cat's-eyeretro-reflectors. A beamsplitter provides that the returning light isalso focused to a similar pattern lying in a plane at or near theviewing area of the disk. Any proper combination of objective lens andtarget will constitute an equivalently effective cat's-eyeretro-reflector. Thus the exact position of the reflected plane patternimaged at or near the viewing area is determined only by the adjustmentof the beamsplitter and various mirrors of the system. This reflectedplane pattern may be thought of as a mapping of the illumination area ofthe disc to the viewing area.

It is apparent that the reflected plane pattern may be out of positionin any of six degrees of freedom. These degrees of freedom are definedas rotation or translation on each of three axes. Accordingly, thecomponent mounting apparatus is provided with six adjustments toaccomplish such rotations and translations. These six adjustments act onthe members employed to direct the reflected plane pattern to theviewing in order to precisely direct the individual disc hole images totheir desired locations. Additionally, the degree of non-interactionbetween adjustment members, the majority being independently referencedto the main frame, results in substantial improvement in ruggedness andtemperature stability. These six adjustments are exclusive of the disccentering adjustments described later.

Referring to the position of the reflected plane pattern or mapping, inthe subsequent description of the embodiment, rotation around either ofthe two axes parallel to the flat surface of the disc (the X and Y axes)is termed keystoning because misadjustment with respect to these axescauses a square pattern of illumination pinpoints to be mapped to theshape of a keystone at the point of intersection of the reflected beamand the disc. Rotation around an axis perpendicular to the disc (the Zaxis) is termed rotation, for purposes of defining the adjustment schemeherein. Translation along the X and Y axes is defined as positionadjustment, and translation on the Z axis perpendicular to the disc istermed size adjustment. Translation on the Z axis also affects the focusof the pattern, but the apparent size of the pattern at the plane of theviewing area is more critical.

In the present invention, keystoning is adjusted by tilting a mirrormounted on the main frame. Position is adjusted by moving the entirescanning disc in either the X or Y direction, again referenced to themain frame. Size and rotation are adjusted by moving a tilt plate onwhich the beamsplitter and one other mirror are mounted. This tilt platecan be translated along the Y axis and tilted on the Y axis.

The alignment process of this invention utilizes cues received fromobserving various light patterns within the microscope and is simplestif keystone, rotation, position, and size adjustments are made in alogical order. The process also relies on certain coarse adjustmentshaving been made during assembly of the microscopes, which adjustmentswould normally not be subject to variation outside the range of theadjustments described by this invention.

The first stage of the alignment process consists of making the keystoneadjustment by removing the objective lens, looking into the lensmounting hole, and observing the reflections of the viewer's eye fromthe surface of the shiny disc used in this invention. Since reflectionsof the eye will be originating in both the illumination area and viewingarea via the beamsplitter, the presence of two images of the viewer'seye indicates the need to tilt the keystone adjusting mirror until theimages are exactly superimposed. None of the subsequent adjustments willaffect the keystone adjustment.

Keystone adjustment is one of the most difficult when the prior artadjustment procedure utilizing the red and green lights is followed.Thus the use of a shiny disk and following the keystone adjustmentprocess of this invention is valuable even if prior art is followed inthe other adjustments.

The remaining adjustments are made with the microscope fully assembledand arranged to observe a high-quality mirror as the target. Again, theobjective and the target mirror act as a cat's-eye reflector. The needfor and degree of adjustment necessary for size, rotation, or positionare determined by observing and interpreting the Moire patterns causedby the pinpoints of reflected light striking the rotating disc andbecoming visible at the eyepiece only when coincident with conjugateholes in the disc. It can be shown that very small offsets in size,position, and rotation of the pattern of reflected pinpoints in anycombination, cause exaggerated movements of the area where pinpoints oflight coincide with their conjugate holes, and thus appear as brightareas in the field of view when viewed through the eyepiece. It is thisexaggerated response that permits easy analysis and precise adjustment.

The various adjustments to be made to achieve size, rotation andposition are made in a specific sequence. The keystoning adjustment mayaffect other adjustments but none of the other adjustments affectskeystoning. Therefore, keystoning is adjusted first. Rotation adjustmentmay affect subsequent adjustments but the size and position adjustmentdo not affect rotation. Therefore, rotation is adjusted second. Sizeadjustment affects only size. Position adjustment affects only positionand precise centering of the disc relative to the shaft does not affectrotations, position keystoning or size of the pattern, that is, itaffects only centering. Size, position, and disk centering are thereforeadjusted last. Making these adjustments in this order ensures that oncean adjustment is made, it need not be remade due to the effects ofsubsequent adjustments. Disc centering is done to meet the requirementthat disc holes in the viewing area be exactly opposite disc holes inthe illumination area where exact opposition is defined relative to theaxis of rotation of the disc.

This process of observing the size and location of bright areas in thefield of view, and of moving various adjusting screws of the independentelements of the apparatus of this invention until desired light patternsare obtained thus makes possible a rugged, stable tandem scanningreflected light microscope having disc holes as small as 20 microns indiameter that is easy to align without use of external light sources,and is capable of unique performance not attainable with other tandemscanning reflected light or other microscopes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a front view of the instrument of this invention showing onlyprinciple optical components.

FIG. 2 is a side view of the instrument of this invention showing onlyprinciple optical components.

FIGS. 3A and 3B are front and side views respectively of the mainframeof the invention.

FIGS. 4A and 4B are front and side views respectively of the mainframeof the invention with the position apparatus and disc added.

FIGS. 5A-5E are various views of the mainframe with the size androtation adjustment apparatus added.

FIG. 6 is a side view of the hub assembly, disc, shaft, bearings, andcompound installed together.

FIG. 7 is a front and cross-section view of the hub assembly and FIG. 7Ais a sectional view along Section Line A--A of FIG. 7.

FIG. 8 is a general layout of the essential elements of the assembledconfocal tandem scanning reflected light microscope.

FIG. 9 illustrates the mapping of the illumination area of the disc ontothe viewing area of the disc when the pinpoints of reflected light areslightly out of adjustment in the X direction.

FIG. 10 illustrates a condition of misalignment where the pattern ofmapped disc holes is in the correct position, but is 5 percent too small(95 percent of the correct size).

FIG. 11 illustrates a condition wherein the pattern of mapped disc holesis 95 percent of correct size and shifted to the right by one holediameter.

FIG. 12 illustrates a condition wherein the pattern of mapped disc holesis in the correct position and of the correct size, but is rotatedclockwise by 2.5 degrees.

FIG. 13 illustrates a condition wherein the pattern of mapped disc holesis of the correct size, is shifted to the right by one hole diameter,and is rotated clockwise by 2.5 degrees.

FIG. 14 illustrates a condition wherein the pattern of mapped disc holesis at 95 percent size, rotated 2.5 degrees, and shifted one holediameter to the right.

FIG. 15 illustrates a condition wherein the pattern of mapped disc holesis at 102 percent of the correct size and shifted one hole diameter tothe right.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT 1. The MountingStructure and Adjusting Apparatuses

Referring to FIGS. 1 and 2 there is illustrated the tandem scanningreflected light microscope of this invention with main light paths andprinciple optical components, without supporting structure and alignmentmeans shown. Light originating from a lamp 5 passes through a condenserlens 11 to a rotating scanning disc 6 into which large numbers of verysmall apertures 7 have been etched or otherwise inserted. Light passingthrough the apertures 7 in the illumination area 8 reflects from mirror1 to mirror 2, reflects from mirror 2 to the beamsplitter 9, andreflects from the partially reflecting surface 14 of the beamsplitter 9to the objective lens 10. The objective lens illuminates the target 15with points of light which are an image of the disc holes 7 in theillumination area 8. Light reflecting from the target 15 is focused bythe objective lens 10, passes through the beamsplitter 9 to mirror 3,reflects from mirror 3 and forms an image at the viewing area 16. Thepoints of light illuminating the target 15 are thus focused onto thedisc holes 7 at the viewing area 16. The light passing through the discholes 7 at the viewing area 16 is then reflected from mirror 4 to arelay lens 17 and an eyepiece 18 where the image is observed, therotation of the disc creating a scanning effect and complete imageformation.

Referring to FIGS. 3A and 3B, there is illustrated the mainframe 19 ofthe support structure of the present invention, comprising a ruggedplate to serve as the reference for all critical elements of themicroscope, and to which are attached fixed 45 degree mirror 3, and a 45degree adjustable mirror 1. Also attached to the mainframe are variousother elements of this invention, including the optical elements, forprecisely adjusting size, rotation, and position of the mapping of thedisc holes so as to complete the desired optical path. These elementsare described in subsequent paragraphs. Mirrors 1, 2, 3, andbeamsplitter 9, and the disk 6 are the elements critical to the properadjustment of the microscope. The exact position of other elements suchas the eyepiece, the objective, and the condenser lens is not criticalto proper alignment. In fact, an alternative system places the objectivein front of the unused side of the beamsplitter. If the described systemis in alignment, the alternative system is also in alignment.

Nominally the centers of mirrors 1, 2, 3, the center of the beamsplitter9 semi-reflecting surface 14, and the optical axis of the objective lensshould all be in a plane parallel to the disc. Also, nominally, theplane of mirror 2 and the beamsplitter semi-reflecting surface 14intersect at right angles defining a line co-linear with the axis ofrotation of the disc. These components may vary from their nominalposition as the microscope is adjusted. In addition, if the mounting ofthese components is not exact, the various adjustments described hereinwill compensate for the errors so that standard art can be used forcomponent mounting.

One easy way to make the shiny disc required by the keystone adjustmentprocedure of this invention is to etch holes in the metal coating of ametal coated glass disc. Prior art is based on the use of a disc made ofsolid opaque material with through-holes etched or otherwise drilled.These discs do not afford the specular reflection required for easykeystone adjustment. When a metal-coated glass disc is used, themetal-coated surface 31 should be facing the light source and eyepiece.The metal-glass interface provides the specular reflections required.The etching of chromium on glass is a common practice.

Referring to FIGS. 4A and 4B, there is illustrated the apparatus foradjusting position, with the scanning disc 6 attached thereto. Theposition adjusting apparatus comprises a compound 20, slidable in the Xand Y axes, by means of screw-adjustable slide pins 21 and 22 andopposing spring-loaded slide pin 23, whose faces conform to the slope ofthe compound to hold the compound in close contact with the mainframe,and to cause very small X and Y movement when the screws 33 and 34 areturned. Additionally, spring-loaded slide pin 23 holds the compound intight contact with slide pins 21 and 22. Slide pins 21 and 22 havespherical surfaces in contact with their adjustment screws 33 and 34,which surfaces minimize twisting torque when the adjusting screws areturned. Pins 21 and 22 must be closely fitted in their respective holesin the mainframe so as to limit their lateral movement as the compoundis adjusted. The scanning disc 6 is centered on a shaft 24, bearings 25,and hub 26 within the compound. Movement of the compound caused byadjusting the slide pins accomplishes the desired X and Y translation ofthe disc in a rugged, stable, and very precise manner, with respect tothe mainframe, and independent of any other adjustments.

The compound 20 has three pads 27 formed on the front surface, whichpads slidably contact the rear surface of the mainframe 19, thusproviding stable three-point support. These pads should have a contactarea with the mainframe 19 of approximately 1/8 square inch each inorder to establish thermal equilibrium between the compound and themainframe. One of these pads 27 should be located directly under each ofthe pins 21, 22, and 23. Additionally, the periphery of the compound isformed conically, resulting in an angled surface for line contact withthe slide pins 21, 22, and 23 at line contact points 28, tightlyconstraining the pads against the mainframe. The total included angle ofthe conical surface should be between 40 and 50 degrees.

There may also be required a keeper slot 29 in the compound, with akeeper screw 30 in the slot in order to prevent excessive rotation ofthe compound 20. As the compound is moved by means of the adjustingscrews 33, and 34, its motion requires that it slide on the face of atleast two of the pins 21, 22, and 23. If the compound is constrained toslide on the face of all three pins 21, 22, and 23 as it is adjusted,the friction will be increased. Screw 30 should be located on a linebetween the face of pin 21, and the faces of pin 22 and slot 29 shouldbe aligned perpendicular to that line, thus allowing the compound 20 torotate about either the face of pin 21 or the face of pin 22 as thecompound is moved. A drive pulley 36 mounted on shaft 24 provides aninterface for rotating the disc. All materials in the mainframe 19, thecompound 20, and slide pins 21, 22, 23 must be of the same or nearly thesame coefficient of thermal expansion to provide thermal stability.

Referring to FIGS. 5A through 5E, there is illustrated the apparatus ofthis invention that permits both size and rotation adjustment. Theapparatus consists of a tilt plate 37, of sufficient thickness andstrength to resist bending, slidably mounted on rods 38 and 39 that arefixed to the mainframe 19 in "V" grooves so as to limit the tilt plateto motion in the Y-direction and rotation around the rods 38 and 39.Rods 38 and 39 are co-linear and their axis intersects the axis ofrotation of the disk. The tilt plate 37 is held against the rods in its"V" notches by screws and springs 43. The axis of the "V" notches in thetilt plate intersects the line of intersection of the planes of surfaces14 and 2. Thus, the intersection of the planes of surfaces 14 and 2 isnot displaced in the X-direction due to thermal expansion or contractionof the tilt plate. The adjustment of the microscope is an order ofmagnitude less sensitive to displacement of the intersection in theY-direction. To the tilt plate 37 are mounted the beamsplitter 9 andfixed 45 degree round mirror 2. The tilt plate 37 is permitted to rotateadjustably around the rods 38 and 39, causing rotation adjustments, suchadjustments being controlled by a screw 40, nut 41, washer 46, and leafspring 42. The bottom surface of nut 41 is spherical, engaging thespherical surface of washer 46, thus allowing full surface contact ofwasher 46 to tilt plate 37 surface during tilt. The area of contactbetween the tilt plate and rod 38 should be separated from the area ofcontact between the tilt plate and rod 39 by some distance to ensure afirm control of the position of the tilt plate. Therefore rods 38 and 39are short and separated by the maximum amount allow by the size of thetilt plate.

As shown in FIGS. 5D and 5E, the beamsplitter 9 is held to the tiltplate by a bar 63 and conical disc spring 64 extending from the 45degree round mirror mount to the top of the beamsplitter. The spring 64is shaped so as to evenly distribute hold-down forces. The mirror mount60 is also of sufficient thickness and strength to resist bending forcesapplied by the arm. The round mirror is mounted to the mirror mount withconventional three-point support provided by screws 61 and springs 62.

To cause size adjustments, movement of the tilt plate along the Y-axisis permitted, controlled by a size adjustment screw 44, set in a blockattached to the mainframe. Spring 45 holds the tilt plate against theadjustment screw 44. Adjustment screw 51, mounted in the same block asadjustment screw 44 is used to temporarily displace the tilt plate (andthe size adjustment) during the alignment process, but is withdrawnafter all adjustments are made.

Referring to FIG. 6, there is illustrated a disc mounting apparatus thatpermits centering of the disc to within 1 micron, with relation to itsaxis of rotation. The apparatus comprises a hub assembly 26 that mountsto the disc 6 and a shaft 24, all of which rotate on ball bearings 25within the compound 20. In order to constrain the disc to lie in a planeperpendicular to the shaft, the flange 56 of the hub assembly is mountedin direct contact with the disc, and a wavy washer 57 is located on theopposite surface of the disc to force the disc against the flange.Various adjusters illustrated in FIG. 7 are incorporated that facilitatefine motion of the disc in a plane perpendicular to the shaft.

Referring to FIG. 7, to move the disc 6 in the X and Y directions asrequired for centering on the shaft, there are provided two taper screws58 and 59. The screws are sufficiently tapered as to provide verysensitive adjustments. They are oriented at 90 degrees and press againstspring pads 65 and 66 in the hub that contact the edges of the centerhole of the disc, thereby causing X or Y movement in very smallincrements as the screws are rotated during the centering procedurelater described. A spring 67 and ball 68, located 135 degrees fromeither taper screw oppose the motion, thus assuring that the disc staysin contact with the spring pads.

Referring to FIG. 8, there is illustrated the general layout of theassembled microscope in partial cross-sectional view. Some principleparts of the microscope described earlier are re-identified in FIG. 8,for clarity. These are the mainframe 19, disc 6, compound 20, objectivelens 10, lamp 5, and eyepiece lens 18. In addition, the parts needed tocomplete a working microscope are shown. A drive motor 70, drive pulley71, and drive belt 72 power and rotate the disc via pulley 36. Amounting plate 73 joins mainframe 19 to a lamphouse 74 and viewing tower75, which in turn supports eyepiece 18, and optionally a video or othertype of camera. The mirror 4 described earlier is not shown in thisview, for clarity. Dustcover 76 protects the optical components. Theadditional components shown in FIG. 8 are well known in the art and arenot further described here. Pulley 36 and hub 26 should be accessible toallow adjusting the disc centering when all essential parts of themicroscope are assembled, pulley 36 being used to rotate the disc byhand as required.

The above described apparatuses, installed on the mainframe with theother noted elements comprise a rugged, stable, easily alignable tandemscanning confocal microscope, wherein the scanning disc holes may be assmall as 20 microns in diameter, when used with a properly centeredscanning disc in conjunction with the alignment method described furtherherein.

In addition the portion of the apparatus containing the beamsplitter 9,the disc assembly, and mirrors 1, 2, and 3 along with the requiredmounting and adjustment mechanisms and disc motor can be used in manyforms. The objective lens or an objective lens turret can be used inproper relation to either externally available side of the beamsplitter.The viewing area and the illumination area may be interchanged.Binocular eyepieces, a film camera, or a television camera may be used.None of these variations will affect the alignment requirements. In facta television camera could be used to carry out most of the adjustmentsas described below.

2. The Microscope Alignment Process

The following paragraphs describe the details of the process by whichadjustment of the individual elements described above is made to achievecomplete, precise alignment of the microscope having disc hole diametersas small as 20-microns. While the previous paragraphs describe thephysical structure and mounting and adjustment embodiments for themicroscope components, in order to best use the embodiments, theoperator must obtain and analyze information provided by the microscopeto ascertain when adjustments are needed, which adjustments are needed,and how much adjustment is needed. This invention illustrates a methodof analyzing reflections from the disc, and Moire patterns appearing atthe eyepiece side of the scanning disc, to guide the alignment process.

Referring to FIGS. 1 and 2, the area 16 of the scanning disc waspreviously identified as the viewing area. Similarly, the area of thedisc 8 was defined as the illumination area. For the purpose ofalignment a flat first surface mirror, (a mirror with the reflectivesurface on the front of the glass) located at or near the focal plane ofthe objective lens, is used as a target. In normal operation of themicroscope, as previously described, light from the illumination source5 is reflected from mirrors 1 and 2, and beamsplitter 9, is imaged ontothe target by the objective lens 10. Points of light thus formed arereflected from the target and imaged by the objective through thebeamsplitter, off mirror 3 and onto the disc at the viewing area. Thus,the illumination area can be said to be mapped to the viewing area. In afully aligned microscope, the points of light from the disc holes at theillumination area would be exactly coincident with the disc holes at theviewing area. This alignment of holes remains even as the disc spins onits axis. The view through the eyepiece would be that of a uniformlyilluminated bright area filling the viewing area.

In a misaligned microscope, the pattern of disc holes mapped to theviewing area may be rotated or translated on any of the three axes dueto misalignment of individual components in the path. Some of suchmapped disc holes may by chance happen to align with other disc holes inthe viewing area. In general when one disc hole is aligned perfectly,other nearby holes will have some degree of overlap due to therepetitious patterns of disc holes used in discs designed for this typeof microscope. Such is the case with the disc described in co-pendingpatent application Ser. No. 07/497,318. The result is that when a brightarea is seen through the eyepiece, it will comprise many disc holes. Theview through the eyepiece may then be that of one or more illuminatedbright areas. These bright areas constitute a Moire pattern.

Many of the bright areas may result from the chance alignment of discholes which would not align in a well-adjusted microscope. One suchbright area will, however, always exist when disc holes are properlyaligned. This one bright area will always comprise many disc holesregardless of the design of the disc hole pattern. The Figures describedbelow illustrate the behavior of this one bright area in response to theadjustment of the microscope. Analysis of this view of the unique Moirepatterns formed when the microscope is focused on a high-quality mirroris a major factor of this invention for adjusting tandem scanningreflected light microscopes.

FIG. 9 shows the condition when the illumination area disc holes are outof adjustment in the X direction. Squares are used to represent the discholes of the illumination area as mapped onto the viewing area andcircles to represent the disc holes of the viewing area of the disc. Thecross represents the center of the field of view. Under this conditionof misalignment, no light would be visible through the eyepiece.

FIG. 10 shows a condition of misalignment wherein the illumination areadisc hole pattern is mapped in the correct position, but is 5 percenttoo small. In this example, the disc holes near the center of the fieldof view are in alignment, but farther from the center the misalignmentbecomes complete. Looking through the eyepiece, one would see a brightarea of light in the center of the field of view, representing the areaof coincidence.

FIG. 11 shows the illumination area mapped onto the viewing area withthe disc hole pattern approximately 5 percent too small and out ofposition to the right by about one hole diameter. Under theseconditions, the bright area is shifted to the right of the center of thefield of view. The shift of the bright area to the right is many timesthe diameter of one hole, an important exaggerated effect which is usednot only to identify the misalignment, but also to indicate very smallchanges as fine adjustments are made; thus, precise alignment becomespossible while observing the movement, size, and position of the brightarea with reference to the center of the field of view.

FIG. 12 shows the illumination area mapped onto the viewing area wherethe disc hole pattern is in the correct position and of the correctsize, but rotated 2.5 degrees clockwise. A bright area in the center ofthe field of view results.

FIG. 13 shows the illumination area mapped onto the viewing area wherethe disc hole pattern is of the correct size, is rotated 2.5 degreesclockwise, and is shifted to the right about one hole diameter. Thebright area is shifted downward from the center of the field of view.

FIG. 14 shows the illumination area mapped onto the viewing area 5percent too small, rotated 2.5 degrees, and shifted to the right by onehole diameter. In this case, the bright area is shifted down and to theright. FIGS. 10, 12, and 13 show how the response of the bright area tothe position adjustment can be used to detect misadjustment of rotation.If the bright area moves exactly in the X direction in response to the Xposition adjustment the rotation adjustment is correct. If the brightarea moves in the Y direction or diagonally when the X positionadjustment is used, then rotation is misadjusted.

FIG. 15 shows the illumination area mapped onto the viewing area 2percent too large and shifted to the right by one hole diameter. In thisfigure the bright area has moved to the left. A comparison of FIGS. 11and 15 shows how the bright area can be used to identify sizemisadjustment. In FIG. 15 the bright area is larger than in FIG. 11indicating that the size misadjustment is greater in the case of FIG. 11than in the case of FIG. 15. In addition, in FIG. 11 the bright areamoves to the right in response to misadjusting the illumination areamapping to the right, indicating that the mapping is too small. In FIG.15 the bright area has moved to the left in response to misadjusting theillumination area mapping to the right, thus indicating that the mappingis too large. In fact the bright area may be larger than the field ofview and the size adjustment still not be perfect. In this case thebright area can still be moved by the position adjustments. If themapping position is moved to the right and the left side of the field ofview dims first, then the bright area is moving to the right and themapping is too small. If under these same conditions the right side ofthe field of view dims first then the mapping is too large. When thesize is in perfect adjustment, the bright area is, in effect, infiniteand the entire field of view will dim uniformly in response to themisadjustment of position, as might be inferred from FIG. 9.

The preceding examples illustrate that by correlating the location andsize of the bright areas appearing on the eyepiece area of the disc, theoperator may determine the degree and type of misalignment of the discholes, and importantly, may accomplish very precise alignment of themicroscope by using the size, rotation, and position adjusting screws ofthis invention. This adjustment procedure is easy to use. The brightareas in the field of view are large, and the motions are large comparedto motions of individual disc holes as mapped. Later paragraphs willdescribe the optimum order in which to make the adjustments.

The description of the Figures given above assumes that there is nomisadjustment of keystone. Analysis of the bright areas can be used toidentify misadjusted keystone, but the analysis is difficult, especiallywhen the keystone misadjustment is combined with other misadjustments. Amuch simpler method for adjusting keystone is here described which isalso a subject of this invention. This method requires that themicroscope use a shiny disc or similar device to provide a specularreflection when viewed from the location of the objective lens.

The microscope has two optical axes. One optical axis is defined by thelight path from the center of the objective lens to the center of theviewing area. The other optical axis is the light path from the centerof the illumination area to the center of the objective lens. Ideallyeach of these axes should intersect the disc at right angles, in whichcase the keystone will be properly adjusted. The keystone will becorrectly adjusted, however, if the two axes intersect the disc atcorrespondingly equal angles; that is, if one axis is tilted 1 degree,then the other should be tilted 1 degree in the opposite directionbecause of the image reversal which takes place in the mapping of theillumination area to the viewing area. Keystone misadjustment isdetected by simultaneously viewing an image reflected from both theviewing area and the illumination area of the disc.

The simplest way to see these reflections is to remove the objectivelens and look in the direction of the beamsplitter through the objectivelens mounting hole. When so doing one can see the image of the viewer'seye reflected from the mirrored surface of the disc in both the viewingarea and the illumination area. If these two images of the eye areexactly superimposed, the keystone is correctly adjusted. Using themirror 1 adjustment screws, the mirror may be tilted until the twoimages coincide. The reflections can be viewed by an alternative means.FIG. 1 shows four sides of the beamsplitter 9. Three sides are used andone side is unused and unobstructed, this latter side being the side ofthe beamsplitter farthest from mirror 2. Thus when the objective lens isremoved, two sides of the beamsplitter are externally available forviewing. The required reflections may be viewed though the unused sideof the beamsplitter and the same adjustments made to the keystone.Alternatively, a lamp or other easily seen target can be placed in frontof one of the externally available beamsplitter sides and its reflectionviewed from the other externally available side. The images of such alamp or target can be seen as reflected from the disc in both theviewing area and the illumination area. Again, mirror 1 is adjusteduntil the two images coincide.

The keystone adjustment is made with the disc spinning as in normaloperation of the microscope so that any tilt of the disc on its shaftwill have no effect on the accuracy of the adjustment. Normally thekeystone adjustment is made before any other adjustment. All otheradjustments can then be made and the correct keystone adjustment willnot be affected.

The centering of the disc is also a critical adjustment easilyaccomplished by observing light patterns through the eyepiece. Theprevious section described a disc hub mechanism permitting centering to1 micron accuracy. The disc centering adjustment is made by firstsetting up the microscope as would be done to accomplish the adjustmentsby Moire patterns, and turning the temporary size adjusting screw 51clockwise to achieve a bright area as indicated in FIG. 11. The discmotor is then turned off. When the disc stops, the individual disc holesof the viewing area can be seen, making the bright area hard to discern.At this point the eyepiece can be defocused, blurring the individualdisc holes until they merge, thus making the bright area easy to seeagain. Disc centering can be tested by rotating the disc by hand andobserving the bright area. If the bright area remains stationary thedisc is centered, but if the bright area moves in an orbit as the discis rotated, the disc is not centered. The disc is centered by adjustingscrews to move the bright area to the center of its orbit. If the discis rotated so that one of the screws is opposite one of the positionadjusting screws, then the two opposite screws have the same effect onthe position of the bright area. Because of the possible diagonal motionillustrated in FIG. 14, it is best to adjust the disc centering afterthe rotation adjustment has been made. Small disc centering errors willnot affect the accuracy of the other adjustments.

It is possible for the disc to be so far off center that the bright areaillustrated in FIG. 10 will actually appear as a bright ring rather thana bright area. This happens if the orbit of the bright area is larger indiameter than the bright area itself. In this case it will be best tomake an approximate adjustment of the disc center before the other Moirepattern adjustments are made. Nonetheless, the keystone adjustment isbest made first.

3. Step-By-Step Alignment Process

In order to demonstrate the application of the invention, the followingparagraphs describe a step-by-step process to achieve alignment easilyand precisely. The process assumes that initial factory alignment, whichis accomplished by aligning certain registration points on the disc, hasbeen accomplished, thus assuring that the process described herein isbegun with a condition of close alignment already existing, as isnormally the case. The method is a follows:

1. Keystone Adjustment

Turn on the disc motor. Remove the objective lens and operate the diskwith the lamp off. Look into the beamsplitter through the objective lenshole at the reflection of your eye. Adjust mirror 1 (FIG. 3) until thetwo reflections of the eye merge into one.

2. Rotation Adjustment

a. Set the microscope up to observe a first surface mirror in the normalmanner of general use of the microscope. Observe through the eyepieceand focus on the mirror while turning the size adjusting screw 44 (FIG.5) clockwise. An area of the field of view will appear bright. Turn thesize adjusting screw clockwise until the bright area decreases in sizeas the screw is turned clockwise. Turn the size adjusting screw untilthe bright area is approximately one third of the diameter of the fieldof view. Some refocusing of the microscope may be required as the sizeadjustment screw is turned.

b. Use the position adjusting screws 33 and 34 on slide pins 21 and 22(FIG. 4) to move the bright area to the center of the field of view.Rotate one of the position adjusting screws and note the direction ofthe motion of the bright area. Nominally the bright area will move inthe direction of action of the position adjusting screw used. If thebright area moves at an angle relative to the direction of action of theposition screw used, the rotation adjusting nut 41 (FIG. 5) should berotated a little at a time until the bright area moves in the nominaldirection in response to the position adjusting screws. In general itwill be necessary to use both position adjusting screws to re-center thebright area as the rotation adjustment is used.

3. Size Adjustment

Re-center the bright area and note the direction of motion of the brightarea in response to the position adjusting screws 33 and 34 on slidepins 21 and 22. Rotate the size adjusting screw 44 counterclockwise andnote that the bright area increases in size. Rotate the size adjustingscrew counterclockwise until the bright area fills the whole field ofview. Continue to rotate the size adjust counterclockwise until thewhole field of view dims uniformly as one of the position adjustingscrews is used to move the bright area. The dimming of one side of thefield of view first implies the motion of the bright area toward theother side. If this direction of motion in response to the positionadjusting screw is opposite to the direction noted, the size adjustment44 must be rotated clockwise until the whole field of view dimsuniformly in response to the position adjusting screw.

4. Position Adjustment

a. Turn the second size adjusting screw 51 (FIG. 5) clockwise until thebright area is slightly smaller than the field of view. Use the positionadjusting screws to re-center the bright area. Rotate the second sizeadjusting screw 51 counterclockwise until the tilt plate 37 (FIG. 5)rests against the first size adjusting screw 44.

b. Position may be adjusted by turning the position adjusting screws soas to achieve the maximum brightness of the field of view.

To achieve the approximate adjustment required prior to beginning thisprocedure, conventional industry adjustment methods can be used.Preceding the approximate adjustment by the procedure of step one willsimplify the process.

5. Disc Centering Process

a. Arrange the microscope to observe a first surface mirror in thenormal manner of general use of the microscope. Focus on the mirrorwhile turning the second size adjusting screw 51 (FIG. 5) clockwiseuntil the bright area is approximately one third of the diameter of thefield of view. Turn off the disk motor. Individual disc holes can now beseen, and the bright area may be hard to perceive. Defocus the eyepieceuntil the individual holes blur to the point of merging. In thiscondition the bright area will again be perceived.

b. Rotate the disk by hand. If the bright area orbits in a circle as thedisk is rotated, then the disk needs to be centered. Use the diskcentering screws 58 and 59 (FIG. 7) to move the bright area to thecenter of its orbit.

c. Return the second size adjusting screw 51 to its former position andrefocus the eyepiece.

The step-by-step process described above is only one specific method forusing the Moire pattern concept to adjust the microscope. For examplethe rotation adjustment can be made by using the rotation adjusting nutto produce the largest bright area possible without turning the sizeadjusting screw. In general the larger the bright area, the closer toproper alignment is the microscope. Also the brighter the center of thefield of view, the closer the alignment is. These generalities can allowthe adjustment of tandem scanning reflected light microscopes ofentirely different design.

Many variations and modifications of the above-described embodiments arewithin the ordinary skill of the skilled artisan in this art, withoutdeparting from the scope of the invention. Accordingly, thosemodifications and embodiments are intended to fall within the scope ofthe invention as defined by the following claims.

I claim:
 1. A tandem scanning confocal reflected light microscope,comprisinga base member, a scanning disc rotatable about an axis, havinga plurality of identical patterns of transparent areas, a plurality ofelements in a light path that directs light from a light source (1)through an illumination region of the transparent apertures of the discto (2) a beamsplitter for directing light passing through the disc to alocation wherein a specimen to be analyzed is to be located, and (3)passing light reflected from a specimen through the beamsplitter to aviewing area on the disc, said viewing area having a pattern oftransparent areas identical to those of the illuminated region, saidscanning disc having planer opposed surfaces and a light reflectivematerial for causing light impinging on said disc to be subjected tospecular reflection so as to produce an image.
 2. A tandem scanningconfocal reflected light microscope according to claim 1 whereinsaiddisc is fabricated from a transparent material, and said lightreflective material is a medium formed as a coating on one of saidsurfaces.
 3. A scanning disc according to claim 2 wherein said lightreflective material is located at the interface of said disc and saidcoating.
 4. A tandem scanning confocal reflected light microscopeaccording to claim 1 wherein said transparent areas are approximately 20to 30 microns in diameter.
 5. A tandem scanning reflected lightmicroscope according to claim 1 further comprisinga beamsplitterconstituting one of said elements in said light path, said beamsplitterbeing located such that said light reflective material reflects lightfrom and toward said beamsplitter.
 6. A tandem scanning confocalreflected light microscope, comprisinga base member, a scanning discrotatable about an axis, having a plurality of transparent areas thatare identically located on opposite sides of the disc, a plurality ofelements in a light path that directs light (1) through an illuminationregion of the transparent apertures of the disc to (2) a region whereina specimen to be analyzed is to be located, and (3) passing lightreflected from a specimen to a viewing area on the disc, said viewingarea having a pattern of transparent areas identical to those of theilluminated region, a tilt plate fixed to said base member, said tiltplate having a beamsplitter and fixed mirror mounted thereon, said tiltplate being constrained in motion in the x-direction along a linepassing approximately through the line of intersection of the plane ofthe semi-reflective surface of the beamsplitter and the plane of thesurface of the fixed mirror.
 7. A tandem scanning confocal reflectedlight microscope, comprisinga scanning disc rotatable about an axis, andhaving a plurality of transparent areas that are identically located onopposite sides of the disc, a base member for supporting said disc and aplurality of elements in a light path that directs light (1) through anillumination region of the transparent apertures of the disc to (2) aregion wherein a specimen to be analyzed is to be located, and (3)passing light reflected from a specimen to a viewing area on the disc,said viewing area having a pattern of transparent areas identical tothose of the illuminated region, a main frame having a large flatsurface, a compound mounted parallel to said large flat surface andhaving a central axis perpendicular to said large flat surface andcollinear with the axis of rotation of said scanning disc, said compoundhaving contact with said large flat surface to maintain the parallelrelationship, means for moving said compound parallel to said large flatsurface in various directions and in rotation relative to said centralaxis, said scanning disc mounted on said compound for rotation about theaxis of said disc.
 8. A tandem scanning confocal reflected lightmicroscope as in claim 7, whereinsaid scanning disc has a reflectivemedium on a surface parallel to said large flat surface.
 9. A tandemscanning confocal reflected light microscope as in claims 7 or 8,whereinsaid elements are independently adjustable to provide sizeadjustments that affect only size and position adjustments that affectonly position.
 10. A tandem scanning confocal reflected light microscopeas in claims 7 or 8, further comprisinga tilt plate mounted on a surfaceof the base member parallel to said large flat surface for rotationabout and translation along an axis in a plane parallel to said largeflat surface, said elements mounted on said tilt plate to control sizeand rotation, adjustment, and means for adjustably rotating said tiltplate about said axis and translating said tilt plate along said axis.11. A tandem scanning confocal reflected light microscope as in claim10, whereinthe materials of said frame, said compound said means formoving said compound, said tilt plate and said means for adjusting allhave substantially the same coefficients of thermal expansion.
 12. Atandem scanning confocal reflected light microscope, comprisingascanning disc rotatable about an axis, and having a plurality oftransparent areas that are identically located on opposite sides of thedisc, a base member for supporting said disc and a plurality of elementsin a light path that directs light (1) through an illumination side ofthe transparent apertures of the disc to (2) a region wherein a specimento be analyzed is to be located, and (3) passing light reflected from aspecimen to a viewing area on the disc,said viewing area having apattern of transparent areas identical to those of the illuminatedregion, a main frame having a large flat surface, a compound mountedparallel to said large flat surface and having a central axisperpendicular to said large flat surface and collinear with the axis ofrotation of said scanning disc, said compound having three points ofcontact with said large flat surface to maintain the parallelrelationship, means for moving said compound parallel to said large flatsurface in various directions and in rotation relative to said centralaxis, said scanning disc mounted on said compound for rotation about theaxis of said disc.